1. Field of the Invention
This invention relates generally to novel methods for enhancing the biological activity of chemotherapeutic agents. More specifically, the invention pertains to unique methods for increasing the toxicity of cytostatic hydrophobic chemotherapeutic agents using multidrug resistant reversing agents in which the multidrug resistant reversing agents are macrocyclic lactone compounds.
2. Description of Related Art
Selection of tumor cells with cytostatic hydrophobic drugs has been shown to result in the development of a multidrug resistance (mdr) phenotype and in the overexpression of P-glycoprotein (Pgp) (Gottesman et al., Biochemistry of multidrug resistance mediated by the multidrug transporter, Annu. Rev. Biochem. 62: 385-427, 1993; Endicott et al., The biochemistry of P-glycoprotein-mediated multidrug resistance, Annu. Rev. Biochem. 58: 137-171, 1989). Pgp is a member of the ABC (ATP binding cassette) superfamily of membrane transporters that includes the multidrug resistance associated protein MRP (Cole et al., Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells, Cancer Res. 54: 5902-5910, 1994), the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al., Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines, Nature 316: 817-819, 1985) and several bacterial periplasmic membrane proteins (Higgins, ABC transporters: from microorganisms to man, Annu. Rev. Cell Biol. 8: 67-113, 1992). Although Pgp has been shown to cause multidrug resistance (MDR) in tumor cells, its function in normal tissues is less certain. Pgp gene family in rodents and humans consists of three classes (I, II, and III) and two classes (I and III), respectively. Moreover, while classes I and II have been shown to cause MDR, class III of both rodents and humans does not. Using homologous recombination, it was shown that class I Pgp is involved in drug transport in normal tissues while class III Pgp mediates phosphatidyl-choline transport (Smit et al., Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease, Cell 75: 451-462, 1993) and may be "a flipase" (Ruetz et al., Phosphatidyl-choline translocase--a physiological role for the mdr2 gene, Cell 77: 1071-1081, 1994).
Various levels of Pgp expression have been shown in tumors from different cancers (Goldstein et al., Expression of a multidrug resistance gene in human cancers, J. Natl. Cancer Inst. 81: 116-124, 1989). However, more studies are needed to determine if changes in Pgp levels in tumor cells are prognostic of MDR. Recently, Pgp expression in sarcomas of children and neuroblastomas was shown to correlate with low response to chemotherapy and the long term survival of patients (Chan et al., Multidrug resistance in pediatric malignancies, Hematol. Oncol. Clin. North Am. 9: 275-318, 1995; Chan et al., P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma, N. Engl. J. Med. 325: 1608-1614, 1991). Other studies using MDR-reversing drugs have implicated Pgp in some MDR cancers (Ford, MODULATORS OF MULTIDRUG RESISTANCE, Preclinical Studies, Hematol. Oncol. Clin. North Am. 9: 337-361, 1995; Ozols, Clinical reversal of drug resistance--foreword, Curr. Probl. Cancer 19: 69-123, 1995; Patel et al., Multidrug resistance in cancer chemotherapy, Invest. New Drugs 12: 1-13, 1994). Nevertheless, a clear clinical benefit of MDR-reversing drugs remains to be demonstrated. Earlier attempts to use verapamil as an MDR-reversing drug have been hampered by its high cardiotoxicity (Dalton et al., A phase III randomized study of oral verapamil as a chemosensitizer to reverse drug resistance in patients with refractory myeloma, Cancer 75: 815-820, 1995; Pennock et al., Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration, J. Natl. Cancer Inst. 83: 105-110, 1991). In tumoral lymphoid (CEM) cells, one study showed a higher retention of cyclosporin A (CsA) in Pgp-lacking parental cells than in Pgp-expressing MDR cells leading the authors to believe that Pgp blockers could restore CsA retention in the MDR-CEM cells (Didier et al., Decreased uptake of cyclosporin A by P-glycoprotein (Pgp) expressing CEM leukemic cells and restoration of normal retention by Pgp blockers, Anti-cancer Drugs, 6: 669-680, 1995). While the results obtained with cyclosporin A and the non-immunosuppressive analog SDZ-PSC 833 have been encouraging, some toxic effects were also observed when cyclosporin A was used in clinical studies (Warner et al., Phase I-II study of vinblastine and oral cyclosporin A in metastatic renal cell carcinoma, Am. J. Clin. Oncol. 18: 251-256, 1995; Murren et al., A phase II trial of cyclosporin A in the treatment of refractory metastatic colorectal cancer, Am. J. Clin. Oncol. 14: 208-210, 1991). Hence, the identification of MDR-reversing drugs with low toxicity to the mammal or human undergoing chemotherapy is a major concern for the clinical treatment of MDR tumors.
Previously, macrocyclic lactone compounds such as the LL-F28249 compounds, the milbemycins and the avermectins have been widely used for treatment of nematode and arthropod parasites. The highly active LL-F28249 family of compounds are natural endectocidal agents isolated from the fermentation broth of Streptomyces cyaneogriseus subsp. noncyanogenus. U.S. Pat. No. 5,106,994 and its continuation U.S. Pat. No. 5,169,956 describe the preparation of the major and minor components, LL-F28249.alpha.-.lambda.. The LL-F28249 family of compounds further includes, but is not limited to, the semisynthetic 23-oxo derivatives and 23-imino derivatives of LL-F28249.alpha.-.lambda. which are shown in U.S. Pat. No. 4,916,154. Moxidectin, chemically known as 23-(O-methyloxime)-LL-F28249.alpha., is a particularly potent 23-imino derivative. Other examples of LL-F28249 derivatives include, but are not limited to, 23-(semicarbazone)-LL-F28249.alpha. and 23-(thiosemicarbazone)-LL-F28249.alpha..
The milbemycins, also known as the B-41 series of antibiotics, are naturally occurring macrocyclic lactones isolated from the microorganism, Streptomyces hygroscopicus subsp. aureolacrimosus. U.S. Pat. No. 3,950,360 shows the preparation of the macrolide antibiotics milbemycin.sub..alpha.1-.alpha.10, milbemycin.sub..beta.1-.beta.3 etc. These compounds are also commonly referred to as milbemycin A, milbemycin B, milbemycin D and the like, or antibiotic B-41A1, antibiotic B-41A3, etc.
The avermectins, also known as the C-076 family of compounds, are naturally occurring macrocyclic lactones produced by the soil actinomycete microorganism, Streptomyces avermitilis. U.S. Pat. No. 4,310,519 discloses the isolation and preparation of the major components A.sub.1a (e.cf avermectin A.sub.1a), A.sub.2a, B.sub.1a and B.sub.2a, and the minor components A.sub.1b (e.c., avermectin A.sub.1b), A.sub.2b, B.sub.1b and B.sub.2b. The C-076 family additionally embraces the semisynthetic derivatives such as the 22,23-dihydroavermectins described in U.S. Pat. No. 4,199,569. The semisynthetic derivatives include, but are not limited to, ivermectin, abamectin, doramectin, eprinomectin and the like.
Ivermectin (IVM), chemically known as 22,23-dihydroavermectin B.sub.1 or 22,23-dihydro C-076 B.sub.1, is shown, for example, to be an anthelmintic of great efficiency and low toxicity (Campbell, Ivermectin and Abamectin, Springer, N.Y., 1989). IVM has been successfully used orally, by subcutaneous injection or transdermal uptake to cure nematode infections in animals and has also been used in humans to treat several types of infections, such as onchocerciaisis (river blindness). Although the molecular mechanism of the antiparasitic effects of IVM is not completely understood, it is thought that IVM binds with high affinity to a glutamate-gated chloride channel in nematodes (Cully et al., Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans, Nature 371: 707-711, 1994). Indeed, IVM is highly selective for the invertebrate chloride channel but binds with only low affinity to the .tau.-aminobutyric acid-gated (GABA-gated) chloride channel in vertebrate brain (Cully et al., Solubilization and characterization of a high affinity ivermectin binding site from Caenorhabditis elegans, Mol. Pharmacol. 40: 326-332, 1991; Schaeffer et al., Avermectin binding in Caenorhabditis elegans. A two-state model for the avermectin binding site, Biochem. Pharmacol. 38: 2329-2338, 1989). The binding of IVM to the invertebrate glutamate-gated chloride channel, which is essentially irreversible, keeps the chloride channel open and prevents membrane depolarization, leading to the paralysis of the nematode. The low host toxicity of IVM is due to both the low affinity towards the host receptor and the compartmentalization of the receptor in the brain. IVM, which is very hydrophobic, does not effectively cross the blood-brain barrier at low concentrations (Chiu et al., Absorption, tissue distribution, and excression of tritium-labeled ivermectin in cattle, sheep and rat, J. Agric. Food. Chem. 38: 2072-2078, 1990).
In a study using transgenic mice that had their Pgp I function disrupted by homologous recombination, IVM accumulation in the brain and in several other organs was increased dramatically (Schinkel et al., Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs, Cell 77: 491-502, 1994). Ivermectin was observed as being toxic (CNS toxicity) in the mice with the knockout P-glycoprotein. The results led the authors of that study to postulate that the Pgp in normal tissues mediates IVM transport. However, no direct biochemical evidence was shown to support their conclusion. Furthermore, these workers did not demonstrate that ivermection was a multidrug resistance reversing agent. It has also been reported that ivermectin was actively transported across the membranes of canine kidney cells and that P-glycoprotein inhibitors such as verapamil and cyclosporin A inhibited this transport by these cells (Greenwald et al., "Mechanistic Studies of Passive and Active Transport Processes in MDCK Cells: Transport of Selected Anthelmintics," proceedings of the 4lst Annual Meeting of the American Association of Veterinary Parasitologists, Jul. 20-23, 1996, Louisville, Ky.).
Additional studies involving ivermectin have shown acute neurotoxicity in normal mice pretreated with SDZ-PSC 833, a blocker of class I mdr gene-encoded Pgp which was developed for reversal of multidrug resistance of tumor cells (Didier et al., Decreased biotolerability for ivermectin and cyclosporin A in mice exposed to potent P-glycoprotein inhibitors, Int. J. Cancer, 63: 263-267, 1995). Recently, it has been suggested that ivermectin may act as a substrate and an inhibitor of Pgp but the study failed to adequately explain the ivermectin toxicity found in SDZ-PSC 833-treated mice (Didier et al., The abamectin derivative ivermectin is a potent P-glycoprotein inhibitor, Anti-Cancer Drugs, 7: 745-751, 1996).